The biocontrol agent formulation is a fundamental step for using these microorganisms in agriculture. Among the advantages of the bioagent formulation, one can cite the prolongation of the shelf-life of the microorganisms, the efficacy, growth and survival thereof in the environment, coupled to the compatibility with cultural practices (MARTIN, F. N.; LOPER, J. E. Soilborne plant disease caused by Pythium spp.; ecology, epidemiology, and prospects for biological control. Critical Reviews in Plant Sciences, v. 18, n 2, p. 111-181, 1999). Different products may be used in the biological-control agent formulation, which are organic or inorganic, as for instance, talc, turf, kaulinite and vermiculite (NAKKEERAN, S.; DILANTHA, W. G. F.; SIDDIQUI, Z. A. Plant growth promoting rhizobacteria formulations and its scope in commercialization for the management of pests and diseases. In. PGPP: Biocontrol and Biofertilization, Ed. Siddiqui, Z. A. p. 257-296, 2005).
The prior art shows that coconut fiber and the Pseudomonas bacterium have already been used separately as a part of the composition in biological control formulations (WO2009135289, US2009274646, JP2008120752, US2002146394, WO200265836, DE29910002, ZA9510628) and biofertilizers (U.S. Pat. No. 7,405,181, NL1005417, GB2252553, FR2722058, CN101624319, CN101497542, CN101468924, CN101284740, CN101054568, RO85118), but none of these documents shows the relationship between the use of coconut fibers and Pseudomona bacterium for biological control of plant diseases, promoting plant growth and, biofertilizing agente and with such efficiency.
Formulations with Gram-positive bacteria, such as those of the genus Bacillus, are more frequent on the market, when compared with Gram-negative bacterium formulations, such as those belonging to the genus Pseudomonas (BETTIOL, W.; MORANDI, M. A. B.; PINTO, Z. V.; PAULA-JÚNIOR, T. J.; CORRÊA, E. B.; MOURA, A. B.; LUCON, C. M. M.; COSTA, J. C. B.; BEZERRA, J. L. Commercial bioprotectors for controlling plant disease—part I. In. Annual Review of Plant Pathology, Ed. Luz, W. C. p. 111-148, 2009). Bacteria of the genus Bacillus produce endospores, resistant structures that guarantee a long shelf-life for the formulated product. As an example of the longer shelf-life of products formulated with the different groups of bacteria, one can cite the 2-year shelf-life of the Companion® product, formulated with Bacillus subtilis GB03, and the 56-day shelf-life of the products Cedomon® and Cerall®, formulated with Pseudomonas chlororaphis, at the same temperature.
Bacteria of the genus Pseudomonas have a high potential for the development of commercial products, due to the high effectiveness in controlling plant disease of the aerial part and of the root system in different types of crops (KHAN, A.; SUTTON, J. C.; GRODZINSKI, B. Effects of Pseudomonas chlororaphis and root rot in peppers grown in small-scale hydroponic troughs. Biocontrol Science and Technology, v. 13, n. 6, p. 615-630, 2003; NAKKEERAN, S., KAVITHA, K., CHANDRASEKAR, G., RENUKADEVI, P., AND FERNANDO, W. G. D. Induction of plant defence compounds by Pseudomonas chlororaphis PA23 and Bacillus subtilis BSCBE4 in controlling damping-off of hot pepper caused by Pythium aphanidermatum. Biocontrol Science Technology, v. 16, p. 403-416, 2006; STOCKWELL, V. O.; STACK, J. P. Using Pseudomonas spp. for integrated biological control. Phytopathology, v. 97, p. 244-249, 2007).
Various action mechanisms for biological control of plant disease have been discovered with the study of this bacterium genus (BAKKER, P. A. H., PIETERSE, C. M. J., AND VAN LOON, L. C. Induced systemic resistance by fluorescent Pseudomonas spp. Phytopathology, v. 97, p. 239-243, 2007; BLOEMBERG, G. V., AND LUGTENBERG, B. J. J. Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Current Opinion of Plant Biology, v. 4, p. 343-350, 2001; KLOEPPER, J. W.; TUZUN, S.; KUC, J. Proposed definitions related to induced disease resistance. Biocontrol Science Technology, v. 2, p. 349-351, 1992). However, there are few studies regarding the Pseudomonas formulation. This fact can be explained due to the sensitivity of this genus to the various conditions and, as a result, and difficult formulation and commercial application (PAULITZ, T. C.; BÉLANGER, R. R. Biological control in greenhouse systems. Annual Review Phytopathology, v. 39, p. 103-133, 2001).
Formulations of species of Pseudomonas with substrates based on talc and turf were suggested by Vidhyasekaran and Muthamilan (VIDHYASEKARAN, P.; MUTHAMILAN, M. Development of formulation of Pseudomonas fluorescens for control of chickpea wilt. Plant Disease, v. 79, p. 782-785, 1995), Krishnamurthy and Gnanamanickan (KRISHNAMURTHY, K., AND GNANAMANICKAN, S. S. Biological control of rice blast by Pseudomonas fluorescens strain Pf714: Evaluation of a marker gene and formulation. Biological control, v. 13, p. 158-165, 1998), Wiyono et al. (WIYONO, S.; SCHULZ, D. F.; WOLF, G. A. Improvement of the formulation and antagonistic activity of Pseudomonas fluorescens B5 through selective additives in the pelleting process. Biological Control, v. 46, p. 348-357, 2008), Kloepper e Schroth (KLOEPPER, J. W.; SCHROTH, M. N. Development of a power formulation of rhizobacteria for inoculation of potato seed pieces. Phytopathology, v. 71, p. 590-592, 1981), Nakkeeran et al. (NAKKEERAN, S., KAVITHA, K., CHANDRASEKAR, G., RENUKADEVI, P., AND FERNANDO, W. G. D. Induction of plant defense compounds by Pseudomonas chlororaphis PA23 and Bacillus subtilis BSCBE4 in controlling damping-off of hot pepper caused by Pythium aphanidermatum. Biocontrol Science Technology, v. 16, p. 403-416, 2006), Vidhyasekaran e Muthamilan (VIDHYASEKARAN, P.; MUTHAMILAN, M. Evaluation of a powder formulation of Pseudomonas fluorescens Pf1 for control of rice sheath blight. Biocontrol Science Technology, v. 9, p. 67-74, 1999) and Vidhyasekaran et al. (VIDHYASEKARAN, P., SETHURAMAN, K., RAJAPPAN, AND VASUMATHI, K. Power formulations of Pseudomonas fluorescens to control pigeonpea wilt. Biological Control, v. 8, p. 166-171, 1997); with shelf-life ranging from 2 months at 4° C. (KLOEPPER, J. W.; SCHROTH, M. N. Development of a power formulation of rhizobacteria for inoculation of potato seed pieces. Phytopathology, v. 71, p. 590-592, 1981) to 12 months at 5° C. (WIYONO, S.; SCHULZ, D. F.; WOLF, G. A. Improvement of the formulation and antagonistic activity of Pseudomonas fluorescens B5 through selective additives in the pelleting process. Biological Control, v. 46, p. 348-357, 2008).
The agroindustrial wastes obtained by processing unripe or ripe coconut have been used in improving fibers as fuel for boilers in the production and carpets and upholstery and in the formulation of substrates for agricultural use. The use of coconut fiber in various industrial segments is an alternative to minimize the environmental impact caused by this solid residue (CEMPRE. Perfil de recicladora de fibras de coco (Profile of coconut fiber recycler) São Paulo, 1998. Reciclagem & Negócio:Fibra-de-coco.35p; ROSA, M. F.; SANTOS, J. S. S.; MONTENEGRO, A. A. T.; ABREU, F. A. P.; ARAÚJO, F. B. S.; NORÕES, E. R. Characterization of the unripe coconut bark powder used as agricultural substrate. Fortaleza: Embrapa Agroindústria Tropical, 2001. 6p. Comunicado Técnico, 5; MOREIRA, M. A., DANTAS, F. M., SANTOS, C. P. D., OLIVEIRA, L. M. D., AND MOURA, L. C. Produção de mudas de pimentão com o use de pó de coco (Production of pimiento by using coconut powder) Rev. Fap., v. 4, p. 19-26, 2008).
Pseudomonas chlororaphis 63-28 is one of the best bacterial isolates used for controlling root rot and promoting growth in domestic cultivation of vegetation in Canada (PAULITZ, T. C.; BÉLANGER, R. R. Biological control in greenhouse systems. Annual Review Phytopathology, v. 39, p. 103-133, 2001), being an efficient agent for biological control and promotion of growth in hydroponics (GAGNÉ, S.; DEHBI, L.; LE QUÉRÉ, D.; CAYER, F.; MORIN, J-L.; LEMAY, R.; FOURNIER, N. Increase of greenhouse tomato fruit yields by plant growth-promoting rhizobacteria (PGPR) inoculated into the peat based growing media. Soil Biology Biochnology, v. 25, p. 269-272, 1993; LIU, W.; SUTTON, J. C.; GRODZINSKI, B.; KLOEPPER, J. W.; REDDY, M. S. Biological control of Pythium root rot of chrysanthemum in small-scale hydroponic units. Phytoparasitica, v. 35, p. 159-178, 2007; OWEN-GOING, T. N.; SUTTON, J. C; GRODZINSKI. Relationship of Pythium isolates and sweet pepper plants in single-plant hydroponic units. Canadian Journal of Plant Pathology, v. 25, p. 155-167, 2003). Besides the efficient competition for space and nutrients with cross-link pathogens, the bacterial isolate produces antibiotics, induces resistance to the plants and produces plant growth hormones (PAULITZ, T. C.; BÉLANGER, R. R. Biological control in greenhouse systems. Annual Review Phytopathology, v. 39, p. 103-133, 2001; ZHENG, J., SUTTON, J. C., AND YU, H I. Interactions among Pythium aphanidermatum roots, root mucilage, and microbial agents in hydroponic cucumbers. Canadian Journal of Plant Pathology, v. 22, p. 368-379, 2000). Due to the effectiveness of Pseudomonas chlororaphis 63-28 as a biological control agent, the project SYNERGIE was implemented in Canada, where one of the purposes was to formulate Pseudomonas chlororaphis 63-28 in turf for use in protected cultures, with shelf-life from six months to one year. The researchers found higher survival of the isolate 63-28 in turf with the moisture of 100-150% (v/v) and lower survival in turf with the moisture of 45% and 25%. However, even in the best units the bacterial population dropped to levels lower than 106 ufc/g after 1 to months (PAULITZ, T. C.; BÉLANGER, R. R. Biological control in greenhouse systems. Annual Review Phytopathology, v. 39, p. 103-133, 2001).
On the international market there the bioproducts Cedomon® e Cerall® (Bioagri, Sweden) are available for the treatment of seeds of oat, barley and wheat, formulated with Pseudomonas chlororaphis. The shelf-life of the bioproducts is of 56 days when they are stored at 4° C.-8° C. and of 21 days at room temperature (LANTMANNEN BIOAGRI: http://www.bioagri.se/pseudomonas_eng.html, access on Sep. 25, 2009). The formulation of the present invention demonstrates that Pseudomonas chlororaphis 63-28 has shelf-life of 224 days at 3±1° C. when formulated on coconut fiber, demonstrating that the coconut fiber is very useful for the formulation with this bacterium.
Barriers to the commercial use of Pseudomonas spp. Include the lack of information about the formulation technology that would optimize the cost of mass production and the application of biocontrol agents (WIYONO, S.; SCHULZ, D. F.; WOLF, G. A. Improvement of the formulation and antagonistic activity of Pseudomonas fluorescens B5 through selective additives in the pelleting process. Biological Control, v. 46, p. 348-357, 2008). The results achieved in developing the formulation with coconut fiber indicate the feasibility of its use for Pseudomonas, formulations due to its high shelf-life.
One of the applications of the present invention lies in the use of the formulation in plant development, mainly in hydroponic crops, with a view to promote plant growth.
Ever since the hydroponic cultivation began to be employed on a commercial scale in the year 1940, it has been growing all ove the world (FURLANI, P. R.; BOLONHEZI, D.; SILVEIRA, L. C. P.; FAQUIN, V. Nutrição mineral de hortaliças, preparo e manejo de soluções nutritivas (=Mineral nutrition of vegetables). Informe Agropecuário, v. 20, n. 200/201, p. 90-98, 1999; FURLANI, P. R. Simpósio IV—Pythium em sistemas hidropônicos—danos e perspectivas para o controle: Principais sistemas hidropônicos em operação no Brasil. Summa Phytopathologica, v. 34, p. 146-147, 2008; STANGHELLINI, M. E.; RASMUSSEN, S. L. Hydroponics a solution for zoosporic pathogens. Plant Disease, v. 78, n. 12, p. 1129-1138, 1994). The increase in hydroponic cultivation is due to the advantages provided in the vegetable production by this system, such as the standardization of the production, anticipation of the crop cycle, reduction in the use of water, efficiency in the use of fertilizers and larger production per area (FURLANI, P. R.; BOLONHEZI, D.; SILVEIRA, L. C. P.; FAQUIN, V. Nutrição mineral de hortaliças, preparo e manejo de soluções nutritivas. Informe Agropecuário, v. 20, n. 200/201, p. 90-98, 1999). These advantages are, to a great extent, responsible for the use of nutrient solution that provides the necessary nutrients, keeping the composition close to the roots and maintaining the adequate concentration of nutrients, besides the control of the pH of solution, keeping the latter at levels suitable for the absorption of nutrients (FAQUIN, V.; FURLANI, P. R. 1999. Cultivo de hortaliças de folhas em hidroponia em ambiente protegido. Informe Agropecuário 20: 99-104; FERNANDES, A. A.; MARTINEZ, H. E. P.; PEREIRA, P. R. G.; FONSECA, M. C. M. Produtividade, acúmulo de nitrato e estado nutricional de cultivares de alface, em hidroponia, em função de fontes de nutrientes. Horticultura Brasileira, v. 20, n. 2, p. 195-200, 2002; MEDEIROS, C. A. B.; ZIEMER, A. H.; DANIELS, J.; PEREIRA, A. S. Produção de sementes pré-básicas de batata em sistemas Hidropônicos. Horticultura Brasileira, Brasília, v. 20, n. 1, p. 110-114, 2002).
Presently, the hydroponic production is concentrated on vegetables and flowers, being employed in producing lettuce, eruca (Eruca sativa), water-cress, wild chicory, cole, chives, celery, tomato, cucumber, pimento, strawberry, tubercles and flowers (FAQUIN, V.; FURLANI, P. R. 1999. Cultivo de hortaliças de folhas em hidroponia em ambiente protegido. Informe Agropecuário 20: 99-104; Chatterton, S.; Sutton, J. C. e Boland, G. J. 2004. Timing Pseudomonas chlororaphis applications to control Pythium aphanidermatum, Pythium dissotocum, and root rot in hydroponic peppers. Biological Control 30: 360-373; PAULITZ, T. C.; ZHOU, T. & RANKIN, L. Selection of rhizosphere bacteria for biological control of Pythium aphanidermatum on hydroponically-grown cucumber. Biological Control, v. 2, p. 226-237. 1992.; MEDEIROS, C. A. B.; ZIEMER, A. H.; DANIELS, J.; PEREIRA, A. S. Produção de sementes pré-básicas de batata em sistemas hidropônicos. Horticultura Brasileira, Brasília, v. 20, n. 1, p. 110-114, 2002). In Brazil, the main crop produced in hydroponics is lettuce, this production being located chiefly close to metropolitan regions.
Since the creation of hydroponic systems, these have been modified for better adaptation to environmental, social and economical conditions of each region, with a view to improve the quality and increase the productivity of crops (ANDRIOLO, J. L.; LUZ, G. L.; GIRALDI, C.; GODOI, R. S.; BARROS, G. T. Cultivo hidropônico da alface empregando substratos: uma alternativa a NFT? Horticultura Brasileira, v. 22, n. 4, p. 794-798, 2004). The main hydroponic systems employed are the nutrient film technique (NFT), deep film technique (DFT) or floating, cultivation in substrate and aeropony. The NFT system is the most widely employed technique in Brazil for cultivating leaf vegetables, wherein the nutrient solution is pumped into the channels and pours by gravity, forming a thin solution film that irrigates the roots. The DFT technique is employed by using a deep film of nutrient solution (5 to 20 cm), the plants being placed on a flat table where the solution circulates by pumping and gravity. The cultivation on substrate is used chiefly for large-size crops like cucumber, pimiento and tomato, wherein the nutrient solution circulates through the substrate, which is generally inert, like sand, expanded clay, vermiculite, rock wool, turf and coconut fiber, returning to the nutrient solution tank. In aeropony cultivation the plant roots remain suspended, receiving water and nutrients by means of sprays (FAQUIN, V.; FURLANI, P. R. 1999). Cultivo de hortaliças de folhas em hidroponia em ambiente protegido. Informe Agropecuário 20:99-104).
Root rot caused by species of Pythium is a serious problem for hydroponic cultivation all over the world. Resistant cultivars are not available for the producer, and there are no registered fungicides for use in hydroponics. The main measure for controlling the disease is to prevent the pathogen from getting into the system. Once the disease has installed itself, the suppression thereof may be carried out by adding biological control agents to the nutrient solution. Besides controlling the disease, the introduction of beneficial microorganisms can promote plant growth, increasing the agriculturist's income. The present invention
At present, the main products studied and used for the formulation of bacteria of the genus Pseudomonas are talc, turf, saw-dust, diatomaceous earth, bentonite, cotton meal, vermiculite and wheat bran. One of the advantages of the present technology lies in using an organic substrate, which is abundant as a waste in Brazil, does not contaminate the soil and does not degrade the environment.